assessment of the microtox bioassay as a preditor for anaerobic bacterial toxicity ·...

ASSESSMENT OF THE MICROTOX BIOASSAY AS A PREDICTOR

FOR ANAEROBIC BACTERIAL TOXICITY

A Thesis Presented

by

DORIS SHEPHERD ATKINSON

Submitted to the Graduate School of the

University of Massachusetts in partial fulfillment

of the requirements for the degree of

MASTER OF SCIENCE

September 1987 ^

Department of Civil Engineering



A Thesis Presented

by

DORIS SHEPHERD ATKINSON

Submitted to the Graduate School of the

University of Massachusetts in partial fulfillment

of the requirements for the degree of

MASTER OF SCIENCE

September 1987




A Thesis by:

Doris Shepherd Atkinson


University of Massachusetts

Amherst, Massachusetts

September 1987

Approved as to style and content

Professor Michael S. SwitzenbaumThesis Committee Chairman

Professor James K. EdzwaldThesis Committee Member

Assistant Professor David A. ReckhowThesis Committee Member

William H. HighterDepartment Head, Civil Engineering

11

ACKNOWLEDGEMENTS

This research has been funded by the Massachusetts Division of

Water Pollution Control (contracts no. 83-31 and 87-01). The author

would also like to thank R. Hickey, J. Robins and D. Wagner for their

technical assistance, and Dr. Michael S. Switzenbaum for his suport and

guidance.

111

ABSTRACT

This thesis addresses the following two questions: 1) can

Microtox, a rapid biological toxicity assay, be used to predict

toxicity to anaerobic methane producing bacteria, and 2) can Microtox

be used to evaluate toxicity removal by anaerobic degredation. To

answer these questions, literature and laboratory data from Microtox

testing and anaerobic toxicity testing were compiled. Microtox tests

were performed according to the manufacturer's directions, and toxicity

to methanogens was evaluated using the anaerobic toxicity assay (ATA)

developed by Owen et_ al. (1979). Additionally, data on hydrogen and

carbon monoxide accumulation in toxified anaerobic reactors were used

as indicators of anaerobic toxicity. The results showed no overall

correlation between Microtox data and ATA data. There was also no

correlation between Microtox and hydrogen and carbon monoxide

accumulations. Laboratory studies on the use of Microtox to evaluate

toxicity removal were discontinued due to high background toxicities.

Microtox therefore does not appear to be useful as a predictor of

anaerobic toxicity nor would it be useful as a tool to evaluate

toxicity removal by anaerobic treatment.

IV

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iii

ABSTRACT iv

TABLE OF CONTENTS v

LIST OF TABLES vii

LIST OF FIGURES viii

CHAPTER

1 INTRODUCTION 1

2 BACKGROUND 6

2.1 Anaerobic Toxicity Testing 6

2.2 Hydrogen and Carbon Monoxide Monitoring 8

2.3 Microtox Testing 9

2.4 Complex Effluents 11

2.5 Toxicity Removal 11

3 EXPERIMENTAL METHODS 13

3.1 Overview 13

3.2 Literature Review 13

3.3 Pure Chemical Toxicity 14

3.3.1 Anaerobic Toxicity Assays 14

3.3.2 Microtox Bioassays 22

3.4 Complex Effluent Toxicity 23

3.5 Hydrogen and Carbon Monoxide Data 25

3.6 Toxicity Reduction 26

4 RESULTS • 27

4.1 Literature Review 27

4.2 Pure Chemical Laboratory Results 31

4.2.1 Anaerobic Toxicity Assays 31

4.2.2 Microtox Bioassays 41

4.2.3 Combined Results 45

4.3 Complex Effluents 45



4.6 Summary of Results 55

5 ' DISCUSSION 59

5.1 Evaluation of Microtox as a Surrogate for ATA's 59

5.2 Complex Waste 67



6 CONCLUSIONS 69

BIBLIOGRAPHY 70

VI

LIST OF TABLES

Page

3-1 Stock Solutions for ATA Medium 17

3-2 ATA Medium Preparation 18

4-1 Relative Toxicity - Literature Survey 28

4-2 Literature Toxicity Values 30

4-3 Reproducibility of ATA Results 37

4-4 Comparison of 20 and 8 Day HRT Medium-Fed Seed 39

4-5 Summary of ATA Results 40

4-6 Reproducibility of Microtox Results 43

4-7 Summary of Microtox Results 44

4-8 Combined Data 46

4-9 Comparison of 5 Minute and 30 Minute Microtox Values 48

4-10 Summary of Complex Effluent Results 53

4-11 Comparison of Medium-Fed and Sludge-Fed Seed 54

4-12 Comparison of Microtox with Hydrogen and Carbon

Monoxide Data 56

4-13 Background Microtox Toxicity 57

5-1 Statistical Summary 65

LIST OF FIGURES

Page

3-1 Anaerobic Transfer Set-Up A 19

3-2 Anaerobic Transfer Set-Up B 20

4-1 Microtox 5EC50 vs. Methane Inhibition - Literature Data 29

4-2 Cumulative Methane Production for Non-Toxified Controls

(Media-Fed Seed) 32

4-3 Comparison of Methane and Total Gas Production

(Non-Toxified Controls) 33

4-4 Chloroform ATA - Methane Production 34

4-5 Chloroform ATA - Total Gas Production 35

4-6 Replicate Controls - Methane Production 38

4-7 Microtox Bioassay - Phenol 42

4-8 Microtox 5EC50 vs. Methane Inhibition - Combined Data 47

4-9 Stirred and Non-Stirred Controls (Sludge-Fed Controls) 50

4-10 Mercuric Chloride ATA - Sludge-Fed Seed 51

5-1 Microtox 5EC50 vs. Methane Inhibition - Organics 61

5-2 Microtox 5EC50 vs. Methane Inhibition - Inorganics 62

5-3 Microtox 5EC50 vs. Methane Inhibition - Priority Pollutants 63

5-4 Microtox 5EC50 vs. Methane Inhibition - Priority Organics 64

Vlll

CHAPTER 1 - INTRODUCTION

Recently anaerobic treatment processes have been receiving

increased attention owing to their advantages over aerobic biological

treatment processes. These advantages include the production of

smaller amounts of waste sludge and the production of methane gas as a

useful by-product.

Anaerobic treatment processes convert complex organic material to

carbon dioxide and methane in the absence of molecular oxygen. This

process occurs through a series of biologically mediated steps, each

involving different groups of microorganisms. The fundemental

microbiological relationships have been investigated and reviewed by a

number of authors (McCarty, 1964; Balch, 1979; Taylor, 1982). In the

first step, polysaccarides are broken down to short chain fatty acids,

acetate, carbon dioxide and hydrogen by fermentative bacteria. Another

group of organisms, the acetogenic bacteria, break down fatty acids to

acetate, carbon dioxide and hydrogen. The methanogenic bacteria then

convert hydrogen and carbon dioxide to methane and also convert acetate

to methane and carbon dioxide. The methanogens are generally slow

growing organisms and represent the rate limiting step in anaerobic

digestion.

There are a number of different process configurations used in

anaerobic treatment, and these have been reviewed in the literature

(Speece, 1983; Switzenbum, 1983). Some of the types include:

completely mixed systems, packed bed reactors, anaerobic filters,

upflow anaerobic sludge blankets, and expanded or fluidized bed

reactors. Recent developments in process technology have focused on

techniques which minimize hydraulic retention times while maximizing

solids retention times. This serves to provide the residence time

necessary for the slow growing methanogens while reducing reactor

volume.

One concern about the use of anaerobic treatment methods is the

reliability of processes for wastes which may contain substances toxic

to the methanogenic microorganisms. Another concern is the possible

adverse or synergistic effects toxicants may have in combination with

other toxicants. Anaerobic reactors which receive wastes containing

more than one toxicant may experience upset conditions when each

toxicant by itself may not cause significant toxicity.

Another interest in anaerobic treatment is as a biological

treatment alternative for the purpose of toxicity removal. While

anaerobic treatment processes are often thought of as a means to reduce

levels of organic material, they also offer the opprtunity for

biodegradation of toxic compounds, and there is a growing body of

literature on toxicity reduction through anaerobic treatment (Healy and

Young, 1979; Bouwer and McCarty, 1983; Boyd .et al.,1983; Speece, 1983).

In response to the concern over the toxic effects of wastes on

anaerobic treatment processes, bioassay methods for measuring toxicity

have been developed. The anaerobic toxicty assay (ATA) was initially

developed by Owen _et_ al_. (1979) and is based on techniques developed by

Hungate (1969) which were modified by Miller and Wolin (1974). It is

currently one of the more widely used methods of determining anaerobic

toxicity. The ATA is a batch method which measures the adverse effect

of a substance or mixture on the rate of methane production from an

easily degraded methanogenic substrate. While this method is simple and

relatively inexpensive, it is time consuming, requiring up to two weeks

before results are available.

Another important development in the monitoring of toxic

conditions in anaerobic treatment processes has been the measurement

of key intermediate gases. Hydrogen and carbon monoxide may accumulate

in toxified reactors, and monitoring of these gases has been proposed

as a basis for an early warning system for anaerobic digester upsets

(Hickey, 1987), While hydrogen and carbon monoxide measurement may

prove useful as a process control technique, it would be useful to have

a quick, inexpensive, screening test for wastes entering a treatment

unit to determine likely toxicity before upset occurs.

The Microtox toxicity analyzer, developed by Beckman Instruments

and currently marketed by Microbics Corporation of Carlsbad, CA.,

provides a means for rapid, inexpensive assessment of toxicity of

aqueous samples. The Microtox system is a relatively inexpensive test

which employs aerobic bioluminescent marine bacteria (Photobacterium

phosphoreum) and can yield reproducible results within one hour. Good

correlations between Microtox and rat, fish, daphnid, shrimp, algal,

and other-aerobic bacterial bioassays have been reported in the

literature (e.g. Dutka and Kwan, 1981; Curtis et a . ,1982; Ribo .and

Kaiser, 1983).

The primary objectives of this research were:

1) to determine whether the Microtox system could be used

as a suitable surrogate for the longer ATA test,

2) to evaluate the interactive toxic effects of several

known toxicants with a complex waste,

3) to compare Microtox response to toxicants to hydrogen

and carbon monoxide accumulations in toxified reactors,

and 4) to determine if Microtox could be used to measure

toxicity reduction by anaerobic biological treatment.

The study was conducted in five phases. In the first phase the

available literature on both anaerobic toxicity and Microtox testing

was reviewed and the reported toxcity levels for chemicals tested by

both sytems compared. In the second phase further laboratory tests

using both toxicty methods were performed. A study of the interactive

effects of toxicants and a complex waste was done in the third phase of

the project. In the fourth phase, Microtox data were compared to

hydrogen and carbon monoxide data, and in the last phase, the

feasibility of using Microtox to evaluate toxicity removal by anaerobic

treatment was investigated.

CHAPTER 2 - BACKGROUND

2.1 ANAEROBIC TOXICITY TESTING

Wastes entering an anaerobic treatment unit may contain materials

which are potentially toxic to the organisms responsible for anaerobic

degradation. There are several different classes of materials or

conditions which may cause digester upset. These include: pH, oxygen

contamination, inorganic toxicants, organic toxicants, and specific

biological inhibitors (Speece and Parkin, 1983). Some examples of

inorganic chemicals which are toxic to methane producing bacteria are:

ammonia, cyanide, sulfide, and heavy metals (Mosey and Hughes, 1975;

Yang et_ al., 1980; Parkin e_t_ aJ .,1983). Potentially toxic organics

include: chloroform, formaldehyde, various phenolics and petrochemicals

(Chou e_t_ al.. ,1978; Yang _et_ al. ,1980; Parkin et_ al.,,1983; Benjamin et

al.,1984). Specific biological inhibitors such as methane analogues

and agricultural antibiotics may also cause toxic effects in anaerobic

reactors (Thiel, 1969; Sykes and Kirsh, 1972; Varrel and Hashimoto,

1982; Jarrel and Hamilton, 1985). While there have been some attempts

to produce predictive models for anaerobic toxicity (Parkin and Speece,

1982), direct laboratory testing is still required to determine the

toxic effects of any given waste.

Over the last twenty or so years a number of different approaches

to anaerobic toxicity testing have been used. Tests may be conducted

under batch or continuous flow conditions. The organisms may be in

suspended culture or attached. The culture used may be a mixed cuture

from a reactor, may be a washed cell suspension prepared from reactor

contents, or may be a pure culture isolated in the laboratory. Tests

may be conducted with non-acclimated organisms or organisms which have

had some previous exposure to the toxicant being evaluated and a

variety of different substrates may be used as the food source. These

test parameters as well as other important factors such as solids

retention time, temperature, pH, osmolarity, the presence or absence of

antagonistic or synergistic substances will all bear on the test

results.

The anaerobic toxicity assay (ATA) is a batch test, which uses

suspended, mixed methanogenic cultures obtained from reseach reactors

(Owen et_ al_., 1979). The organisms are fed acetate and propionate as an

easily degraded methanogenic substrate. The toxic substance to be

tested may be either a pure substance or a complex mixture. While the

test was designed to be used with non-acclimated organisms, it can be

adapted to acclimation studies. The test is usually run under

quiescent conditions, but continuous stirring may be used by adding

magnetic stirrer bars to individual serum bottles. The pH of the test

environment is held at or near 7.2 by means of a bicarbonate buffer.

The incubation temperature is 35 C. The parameter measured in the

ATA test is either the rate of total gas production or the rate of

methane production. The volume of gas produced is measured using a

lubricated glass syringe. The suggested means of reporting data is to

present the maximum rate ratio (MRR) which is the maximum rate of gas

production of the toxified sample normalized to that of a non-toxified

control.

The ATA is a simple, inexpensive test, which has advantages over

more complicated continuous toxicity tests. The ATA test is a

practical test which has been used sucessfully by a number of

investigators to test a variety of substances such as pulp mill wastes

(Benjamin et al., 1984), various priority organic pollutants (Johnson

and Young, 1983), and industrial toxicants (Parkin et al.,1983).

However, it is a time consuming test (requiring up to two weeks before

test results are available) and is therefore not as practical for

applications such as influent monitoring.

2.2 HYDROGEN AND CARBON MONOXIDE MONITORING

There are three key steps in the degradation of complex organic

wastes to methane and carbon dioxide. In the first step, fermentative

bacteria break down polysaccarides to short chain fatty acids, carbon

dioxide and hydrogen. In the second step the fatty acids are further

broken down to acetate, carbon dioxide and hydrogen. Methanogenic

bacteria carry out the third step, which is to covert both acetate and

carbon dioxide and hydrogen to methane (McCarty, 1964 ; Balch et

al.,1979). In a well operating system, hydrogen should not accumulate,

but should be used to produce methane as rapidly as it is formed. Its

accumulation in a reactor is an indication of upset conditions (Hickey,

1987). Carbon monoxide has also been found to accumulate under certain

types of upset conditions, especially those caused by the presence of

toxicants (Hickey, 1987). Measurement of these gases can be used as an

early warning of reactor upset conditons.

2.3 MICROTOX TESTING

The Microtox test was originally developed by Beckman Instruments,

but is now marketed by Microbics Corporation of Carlsbad, California.

The Microtox test is a quick, relatively inexpensive bioassay which

uses the aerobic, bioluminescent marine bacterium Photobacterium

phosphoreum. The light emitting biochemical pathway used by the

Microtox organism is known to be an electron transport pathway,

providing the organism with an alternative to cytochrome electron

transport. This pathway is thought to provide the organism with an

adaptive advantage under microaerophilic conditions (Hastings and

Nealson,1977; Hastings _et a ., 1985).

The principle of the Microtox test is that the intensity of

bioluminescence is diminished in response to exposure to toxicants.

This response is generally linear over some range of toxicant

concentration. The light output at specified temperature, pH, and

salinity is easily measured with the Microtox analyzer, which contains

incubation wells, temperature controls and a photomultiplier tube

connected to a digital output display. The analyzer can also be

10

connected to a strip chart recorder for a permanent record of the test

data. Once the bacteria have been prepared for the test procedure, the

initial light level is measured, a toxic challenge added, and the light

level measured again after a specified period of exposure. Generally

the concentration of toxicant causing a 50% decrease in light output

relative to a reagent blank is the reported result (Beckman, 1982).

The Microtox test is a versatile test, which may be used to

evaluate almost any aqueous sample. The test is short (one to two

hours) and requires no laboratory culturing of test organisms. (The

manufacturer supplies standard cultures of lyophilized bacteria in easy

to handle single test reagent vials). It also has good

reproducibility, with a coefficient of variation of 15 to 20% (Bulich

£t £LL., 1981). The Microtox system previously has been used for

effluent monitoring ( Bulich e± al., 1981; Peltier and Weber, 1980;

Samak and Noiseux, 1980; Vasseur £t _al.,1984) as well as wastewater

treatability monitoring (Slattery,1985), studies of toxicity removal in

activated sludge treatment plants (Neiheisel _e_t al. ,1982), evaluation

of fossil fuel process waters (Lebsack et_ al.., 1981), landfill leachate

studies (Sheehan et^ al_. ,1984) and sediment toxicity studies (Atkinson

et al.,1985).

Good correlations between Microtox tests and a number of other

bioassays using organisms from a wide variety of phylogenetic groups

have been found. Chang eit_ al . (1981) found correlation coefficients

(r) of 0.9 and 1.0 between Microtox results and rat and fish LD50's

11

respectivily for typical organic toxicants. Curtis et_ al.. (1982) and

Indorato et_ al. (1983) found correlation coeficients between Microtox

and different species of fish between 0.80 and 0.95. Looking at 20

chlorophenols, 12 chlorobenzenes and 13 para-substituted phenols, Ribo

and Kaiser (1983) compared results of 7 different bioassays with

Microtox results. Two bacterial assays, three fish assays, and Daphnia

and shrimp assays were included. Correlation coefficients for these

studies all fell between 0.82 and 0.96. No examples of Microtox

comparisons to anaerobic systems were found in the literature.

2.4 COMPLEX EFFLUENTS

When a biological system encounters more than one toxicant at a

time, the response to those toxicants may be greater than or less than

the sum of the observed effects of the toxicants acting individually.

When the effects are greater than additive effects, the toxicants are

said to act synergistically, and when the effects are less than

additive effects, they act antagonistically. Complex effluents, such

as pulp mill wastes may contain a wide variety of toxicants. Wastes

with multiple toxicants pose a treatment problem in predicting the

response to those toxicants even when the response to individual

components may be known.

2.5 TOXICITY REMOVAL

Anaerobic treatment as a means to degrade toxic compounds has

12

recently been receiving greater attention. Several papers have been

published presenting methods for determining anaerobic biodegradation

potential (Shelton and Tiedje, 1984 ; Healy and Young, 1979; Owen et

al., 1979). In general, these tests rely on measurement of gas

production from the mineralization of organic pollutants. It should

not be assumed, however, that these tests necessarily measure removal

of toxicity. Intermediates formed during biological degradation may be

as toxic, or in some cases more toxic than the compound from which they

are derived (Bower and McCarty, 1983; Vogel and McCarty, 1985). Thus,

some form of biological evaluation of anaerobic toxicity removal is

warranted.

CHAPTER 3 - EXPERIMENTAL METHODS

3.1 OVERVIEW

The study was divided into 5 phases. In the first phase the

available literature was searched for information on the toxicity of

pure compounds to anaerobic methane-producing bacteria and to the

Microtox organism. During the second stage further data on the

toxicity of pure compounds were obtained in the laboratory using the

Microtox bioassay and the anaerobic toxicty assay (ATA) with media-fed

seed. The third phase was to study the toxic effects of a complex

effluent using ATA's with sludge-fed seed and to compare the response

to toxic chemicals of this seed to that of the media-fed seed. In the

fourth stage Microtox data were compared to data on hydrogen and

carbon monoxide accumulations in toxified anaerobic reactors. During

the fifth stage of the study the feasibility of using the Microtox

bioassay as an indicator of toxicity reduction by anaerobic treatment

was briefly investigated.

3.2 LITERATURE REVIEW

Available literature was searched for information on the toxicity

of pure chemicals to both Microtox and to anaerobic methane-producing

bacteria. Side by side alphabetical lists by chemical of Microtox data

and methanogen data were compiled. An initial assessment of the data

was made by determining which chemicals were more toxic 'to Microtox,

13

14

which were more toxic to methanogens and which were essentially equally

toxic to both. For chemicals for which appropriate data were found,

the concentration causing 50% inhibition of methane production was

plotted against the concentration causing 50% inhibition in the

Microtox bioassay, and statistical relationships determined.

3.3 PURE CHEMICAL TOXICITY

In this phase of the study, additional information for the

comparison of toxicity of chemicals to the Microtox bioassay and

methanogens was obtained in the laboratory. Anaerobic toxicity assays

(ATA's) as developed by Owen et al. (1979), as well as Microtox

bioassays as developed by Beckman Instruments, were performed. In

order to maximize data for comparison, tests were done with chemicals

for which literature information on one or the other bioassay was

available. As more literature data were available for Microtox tests,

this study focused more heavily on anaerobic, toxicity assays. Testing

was also done for selected chemicals to assure that toxicity values

generated in this laboratory were comparable to those found in the

literature.

3.3.1 Anaerobic Toxicity Assays

Anaerobic toxicity assays (ATA's) as developed .and described by

Owen et_ al_. (1979) were performed to determine the toxicity of various

chemicals to methane-producing bacteria. This toxicity assay is a

15

batch test in which the inhibition of methane production from a readily

digested substrate due to a toxicant is measured. An inoculum, or

seed, of bacteria from a methane-producing reactor is added to

anaerobically sealed serum bottles containing medium, substrate and

toxicant. The bottles are incubated, and the gas production monitored

over a period of two weeks.

The anaerobic seed for this phase of the study was obtained from a

15 L mixed culture anaerobic research reactor operated at 20 days solid

retention time (SRT). The reactor was fed a sucrose based medium,

containing bicarbonate buffer, nutrient salts and yeast extract. One

test was also made to compare the seed from the 20 day SRT reactor to

seed from an 8 day SRT reactor maintained on the same feed. The

initial inoculum for starting both the 20 day and 8 day SRT reactors

came from a mixture of several sources, including a dairy manure

digester, a sewage digester and research reactors. Both reactors had

been operating for approximately 15 months prior to this study. Gas

production from each reactor was approximately 50% methane and 50%

carbon dioxide, and both were maintaned at or near pH 7.0. A

preliminary test was made to determine that 30% seed was an appropriate

seed concentration to use for the anaerobic toxicity assays.

The defined medium used for the ATA's contains vitamins, nutrient

salts, bicarbonate, sodium sulfide as a reducing agent, and resazurin

to detect oxygen contamination. The medium and the method of

preparation were the same as those described by Owen _et_ _al^. (1979) with

16

minor modifications. Table 3-1 lists the stock solutions used and

Table 3-2 lists the method of medium preparation. The medium was

prepared fresh at the beginning of each ATA run from stored stock

solutions.

125 mL glass serum bottles (actual volume approximately 164 mL)

were used as the test vessels. All glassware was detergent washed,

rinsed 5 times in tap water, soaked 5-10 minutes in 10% HC1, rinsed 5

times in distilled water and oven dried. Serum bottles were pre-purged

with nitrogen gas then purged with a mixture of 70% nitrogen and 30%

carbon dioxide gas.

ATA medium and seed were transferred to the serum bottles

anaerobically. Volumes of 35 mL medium and 15 mL (30%) seed were used.

The anaerobic transfers were accomplished using one of two different

set-ups. The first set-up is descibed by Owen et_ _al/ (1979), and is

depicted in Figure 3-1. After initial gassing, medium is pipetted by

opening and closing pinch valves VI and V2, while flushing with gas.

After filling, serum caps are inserted while similtaneously removing

the gas flushing needle. This first set-up proved to be somewhat

awkward, and a second set-up was devised which proved to be equally

reliable. This set-up is depicted in figure 3-2. The draw tube of a

manual repipettor is inserted into the medium or seed bottle. Purge

lines are inserted into the bottle and onto the suction vent of the

repipettor. The repipettor is set to the desired volume, and medium or

seed is pipetted into the serum bottles while flushing with the gassing

17

TABLE 3-1 STOCK SOLUTIONS FOR ATA MEDIUM *

Solution Compound Concentration (g/L)

1 Resazurin 1.0

2 (NH4)2HP04 26.7

CaCl2-2H20 16.7NH4C1 26.6MgCl2'6H20 120.0KC1 86.7MnCl2'4H20 1.33CoCl2'2H20 2.0H3B03 0.38

0.180.17

ZnCl2 0.14

370

5 Na2S'9H20 5006 Biotin 0.02

Folic acid 0.02Pyridoxine hydrochloride 0.1Riboflavin 0.05Thiamin 0.05Nicotinic acid 0.05Pantothenic acid 0.05612 0.001p-Arainobenzoic acid 0.05Thioctic acid 0.05

adapted from Owen et al. 1979

18

TABLE 3-2 ATA MEDIUM PREPARATION *

Step Instruction

Add 1 liter distilled water to 2 litervolumetric flask

Add the following:1.8 mL solution 15.4 mL solution 227 mL solution 3

Add distilled water to 1,800 mL mark

Boil for 20 min while flushing withN2 gas

Cool to room temperature(continue flushing with

Add the following:1.8 mL solution 61.8 mL solution 41.8 mL solution 5

Change gas to 30% C02 :70% N2

Add 8.4 g NaHC03 as powder

Continue flushing with 30% C02: 70% N2until dipensed into serum bottles.

adapted from Owen et al. 1979

19

Open-

tipped

Pipet

Defined

Medium

Magnetic

Stirrer

Flushing

Needles

70 % N2

Serum

Bottle

Water

Seal

FIGURE 3-1 ANAEROBIC TRANSFER SET-UP A

(adapted from Owen et al.,1979)

20

70%

Manual

Repipettor

Magnetic

Stirrer

FIGURE 3-2 ANAEROBIC TRANSFER SET-UP B

21

needle. The serum bottles are then stoppered.

After filling with medium and seed, crimped aluminum seals were

placed on the stoppered serum bottles, and the bottles allowed to

equilibrate to 35° C in an incubator. After equilibration, a substrate

spike and varying amounts of toxicant were added to the serum bottles

via syringe. The substrate spike contained 75 rag sodium acetate and

26.5 mg propionic acid sodium salt in a 2 mL volume of distilled water.

Toxicants were dissolved in distilled water, and varying volumes, up to

2 mL, were added to achieve the desired final toxicant concentrations.

In general, controls containing substrate but no toxicant were run in

triplicate, and duplicates of each toxicant concentration were made.

Also, for each run, at least one control without substrate spike was

prepared to assure that excessive methanogenic substrate was not

carried over into the experiment from the anaerobic reactors. .

Once the substrate spike and toxicant had been added, the head

space pressure of each serum bottle was equilibrated to atmosphereic

pressure through a well lubricated glass syringe. Gas inside the

bottle is allowed to expand and push the plunger of the syringe outward

until equilibrium is reached with atraosphereic pressure on the outside

of the syringe. The syringe is then withdrawn and the gas within

discarded. After zeroing, the serum bottles were incubated at 35° C

+/- 1.5° C in a Thelco Precision incubator.

For each ATA run, total gas production as well as methane

production was monitored on a periodic basis for up to two weeks.

22

Total gas production was measured using the glass syringe method. The

percentage of methane in the head space gas at each sampling time was

determined by gas chromatography. 1 mL samples of the head space gases

were injected into a Gow Mac thermal conductivity gas chromatograph,

using helium as the carrier gas and operating at a detector temperature

of approximately 70° C. A calibrated Hewlett-Packard integrator,

connected to the gas chromatograph, provided the output data of methane

as a percentage of the injected sample. The incremental methane

production was calculated as the measured percentage of methane times

the volume of total gas produced plus the change in the stored volume

of methane in the head space of the serum bottles. Cumulative total

gas production and cumulative methane production for controls and each

toxicant concentration were plotted, and the maximum rates of gas

production at each concentration calculated. The ratio of the maximum

rate of gas production to the maximum rate of gas production of the

controls was calculated for each toxicant concentration. The maximum

rate ratios (MRRf s) were used to determine the concentration of

toxicant causing 50% inhibition of the maximum rate of gas production

relative to the controls (MRR = 0.5). These values were then compared

to the concentrations of toxicant causing 50% inhibition in the

Microtox bioassay.

3.3.2 Microtox Bioassays

Microtox toxicity assays were performed following the procedures

23

described by the manufacturer (Beckman, 1982). The Microtox system

employs lyophilized aerobic marine bacteria which, upon reconstitution,

emit light. The initial light level is measured; a toxic challenge is

added and the percent light loss due to the toxicant is determined. Up

to four concentrations may be tested at once.

Microtox reagent (lyophilized bacteria) supplied by the

manufacturer, was reconstituted with a buffered saline reconstitution

solution (also supplied by the manufacturer). The reconstituted

bacteria were allowed to equilibrate for at least 15 minutes at the

test temperature (15° C), after which 10 microliter aliquots were

pipetted into 10 vials, each containing 0.5 mL of 2.2% saline dilution

fluid. After 15-20 minutes the initial light level of each vial was

measured and recorded. Then either 0.5 mL dilution fluid (blanks) or

0.5 mL of one of four serial dilutions of toxicant in dilution fluid

was added to each of 2 of the 10 test vials. After 5 minutes and after

30 minutes exposure time the light level of each vial was again

measured, and the percent light loss relative to the average of the

reagent blanks was calculated. The percent light loss was plotted

against toxicant concentration, and the concentration resulting in 50%

light loss determined by interpolation. This value is referred to as

the 5 minute or 30 minute 50% effective concentration (5EC50 or

30EC50).

3.4 COMPLEX EFFLUENT TOXICITY

24

The toxic effects of a complex effluent and its interactive

effects with various toxicants were studied using anarobic toxicity

assays as previously described. ATA's of a pulp mill waste (PMW)

alone, PMW combined with various toxicants, and toxicants in distilled

water were performed. A different seed inoculum was used in this phase

of the study (sludge-fed seed), and comparisons of the toxicity of pure

chemicals to this seed and to the medium-fed seed used earlier were

made.

The seed used for this phase of the study came from a laboratory

anaerobic sludge digester. This reactor was fed a mixture of waste

activated sludge (WAS) brought to 1.5% total solids with dissolved air

floats (DAF). The initial inoculum for this reactor came from a

municipal anaerobic sludge digester. The laboratory reactor was

operated at 20 days SRT, produced approximately 67% methane and 26%

carbon dioxide, and had been in operation for approximately 18 months

prior to this study. An initial study indicated that a 38% seed

concentration would be appropriate for toxicity testing with the ATA.

The complex effluent used was obtained from the waste stream of an

integrated pulp mill plant. The waste stream contained combined wastes

from the main pulping and milling operations as well as waste from

bleaching operations. Portions of the same effluent sample were used

for each ATA run. A preliminary test of the PMW was made to determine

an appropriate concentration to use for the combined chemical plus PMW

toxicity tests. A final concentration of 3.8% PMW by volume was used

25

for each of the combined tests.

The inhibition caused by toxicant plus PMW was compared to that

caused by toxicant alone and PMW alone. The interactive effects were

determined by assessing whether toxicity due to the mixture was greater

than or less than the toxicity predicted by simple additive effects of

the PMW and the toxicant. Finally, toxicity of pure compounds to the

sludge-fed seed was compared to toxicity to the medium-fed seed which

had been determined earlier.

3.5 HYDROGEN AND CARBON MONOXIDE DATA

Certain toxic upset conditions in anarobic digesters are

characterized by accumulations of hydrogen and carbon monoxide gas. In

this phase of the study, Microtox data were compared to hydrogen and

carbon monoxide accumulations in toxified reactors to determine if

Microtox could be used as a predictor of these upset conditions.

, Microtox data were taken from the literature and previously

gathered laboratory results. Hydrogen and carbon monoxide data were

taken from a study conducted at the University of Massachusetts,

Amherst, in which toxicity assays similar to the ATA's described in

this report were performd (Rickey, 1987). In these studies, waste

activated sludge, seed from sludge-fed digesters and toxicant were

combined in closed serum bottles, and hydrogen and carbon monoxide as

well as methane and carbon dioxide measured over one day. Gas

measurements were made with a trace analytical mecury reduction

26

detector gas chromatograph,

3.6 TOXICITY REDUCTION

The feasibility of using the Microtox bioassay as a measure of

toxicity removal by anaerobic treatment was investigated. It was

proposed to measure the toxicity of compounds to the Microtox organism

both before and after treatment by anaerobic biodegradation in batch

mode. Microtox bioassays were performed on the ATA defined medium and

supernatant from both medium-fed and sludge-fed seed. Supernatant from

the anaerobic seed souces was obtained by centrifugation in a table-top

centrifuge for approximately 10 minutes. Dissolved oxygen measurements

were made using an electronic dissolved oxygen probe in order to assure

that the Microtox tests were not oxygen limited. Additionally,

Microtox tests of air-purged supernatant from the sludge-fed seed and

supernatant of the sludge used to feed this reactor were made. After

reviewing the results of these initial tests, it was decided to

discontinue work on this phase of the study.

CHAPTER 4 - RESULTS

4.1 LITERATURE REVIEW

Table 4-1 presents the results of the initial literature review.

Literature data for both Microtox and anaerobe toxicity were found for

a total of 39 chemicals. Of these, 24 (62%) were found to be more

toxic to Microtox. Eleven (28%) were of the same order of magnitude

toxicity to both Microtox and methanogens, and the remaining 4 (10%)

were more toxic to methanogens. Considerably more data are available

for Microtox testing than for anaerobe testing, and are reported in a

more consistant manner. Many different types of anaerobic toxicity

data were included in the initial review. Some of the data are based

on mixed culture studies while others are based on enriched or pure

cultures of methanogens. Many procedural differences were found in the

anaerobic literature. For some chemicals, reports were found simply

stating that a certain chemical was not toxic at a given concentration.

When this value was appreciably above the reported Microtox 5EC50, the

chemical was listed as more toxic to Microtox.

For chemicals for which there were more detailed literature data,

the concentration of toxicant causing 50% anaerobic inhibition was

determined. The reported values, or in some cases estimates, were

plotted against the Microtox 5EC50 values on logarithmic axes. This

information is presented graphically in Figure 4-1 and in tabular form

in Table 4-2. In cases where more than one Microtox value was

27

28

TABLE 4-1 RELATIVE TOXICITY - LITERATURE SURVEY

More Toxic to Microtox Equally Toxic(Same Order ofMagnitude)

More Toxic toMethanogens

AcroleinAmmonia (Free)Arochor 1242Benzyl AlcoholCatechol4-Chloro-3-methylphenol2-Chlorophenolp-CresolCyanide2,4-Dicholorophenol2,4-DimethylphenolDimethylphthalate2,4-DinitrophenolFormaldehydeMercuric Chloridem-MethoxyphenolNaphthalene1-OctanolPentachlorophenolPhenolResorcinol1,1,2,2-Tetrachloroethane2,4,6-TrichlorophenolZinc Sulfate

Ammonium (Total)CadmiumCarbon tetrachlorideCopperm-CresolEthylacetateNickel ChlorideNitriteNitrobenzene4-Nitrophenol2-Propanol

AcrylonitrileChloroform1,2-DichloroethaneMethanol

29

oo 4 -

aoh- 1 i&-. 3 -i— *CQhHX

, 1 H * l

w2

rc£d 1 -E:

o^ _o 0

3

D

a

a /'aa

^^>1*1 f—*

° n'a . /Q a*a

*

/ a/,--'"°

- 1 0 1 2 3

LOG MICROTOX 5EC50 (mg/L)

FIGURE 4-1 MICROTOX 5EC50 vs. METHANE INHIBITION

LITERATURE DATA

30

TABLE 4-2 LITERATURE TOXICITY VALUES

Chemical Anaerobe 50% MicrotoxInhibition (mg/L) 5EC50 (mg/L)

Acrolein

Acrylonitrile

Ammonia (Free)

Ammonium (Total)

Carbon Tetrachloride

Catechol

Ethyl acetate

Formaldehyde

Nickel Chloride

Nitrobenzene

Pentachlorophenol

2-Propanol

Resorcinol

Zinc Sulfate

11.2

212

59

750

2.2

6,966

969

200

300

12.3

0.5

5,408

3,193

400

0.67

3,910

1.56

4,833

5.6

32

1,180

9.1

410

46.2

0.08

17,700

310

29.7

31

available, the average value was plotted. Statistical analysis of the

data yielded a correlation coefficient of 0.74. The solid line shown

on Figure 4-1 is the line of equal value. Ten out of 14 data points

(71%) lie on or above this line. These represent chemicals which are

either more toxic or equally toxic to Microtox.

4.2 PURE CHEMICAL LABORATORY RESULTS

4.2.1 Anaerobic Toxicity Assays

An initial test was made to determine an appropriate concentration

of medium-fed seed to use in the ATA's. Four seed concentrations were

tested. Figure 4-2 shows the cumulative methane production for

non-toxified controls of each seed concentration. Based on this test

it was decided to use 30% seed for the various ATA's using medium-fed

seed. The production of methane was compared to the production of

total gas. Figure 4-3 shows that for the controls, methane and total

gas production were very similar, but that at later points total gas

production did not level off as flatly as did methane production.

The results of a typical ATA run are shown graphically in Figures

4-4 and 4-5. Figure 4-4 shows the cumulative methane production for

controls and three concentrations of chloroform. This is very similar

to the pattern of total gas production shown in Figure 4-5. The higher

initial gas reading for total gas is most likely due to improper

temperature equilibration prior to zeroing the serum bottles. These

32

so r

40 -

30 -

20 -

10 -

200

FIGURE 4-2 CUMULATIVE METHANE PRODUCTION FOR NON-TOXIFIED

CONTROLS (Medium-Fed Seed)

33

50

40

30

20

10

A Total Gas

A Methane

(30% Medium-Fed Seed)

200

FIGURE 4-3 COMPARISON OF METHANE AND TOTAL GAS PRODUCTION

(Non-Toxified Controls)

50

40

30

20

10

V Control

O 0.25 mg/L Chloroform

• 0.63 mg/L Chloroform

A 1.25 mg/L Chloroform

(Medium-Fed Seed)

50 100

TIME hrs

150 200

FIGURE 4-4 CHLOROFORM ATA - METHANE PRODUCTION

35

T0TAL

GAS

m1s

50

40

30

20

10

V Control

O 0.25 mg/L Chloroform

• 0.63 mg/L Chloroform

A 1.25 mg/L Chloroform

(Medium-Fed Seed)

50 100 150

TIME hrs

200

FIGURE 4-5 CHLOROFORM ATA - TOTAL GAS PRODUCTION

36

results are used to calculate the concentration of toxicant which will

result in a 50% inhibition of the maximum rate of methane or total gas

production.

In order to assure that our laboratory results could be compared

reasonably to results reported in the literature, ATA assays were

performed using chemicals for which there were previously reported

literature data. Also, duplicate ATA's of two chemicals were run.

Table 4-3 presents these results. The reproducibility between

laboratories appears to be exceptionally good (1 and 5.5% differences

in values). Intra-laboratory reproducibility, however, showed much

higher differences in values (28 and 34% differences). Replicates of

controls and toxified samples were generally well matched. Figure 4-6

shows the cumulative methane production for three replicate controls.

The coefficient of variation between the three replicates (standard

deviation / mean) averages around 3.1% for measurements at different

time points.

A brief study was made to investigate potential differences in

toxicity of chemicals to two anaerobic seeds from reactors which

differed only in their solids retention times. Table 4-4 presents the

comparison of toxicity to 20 day and 8 day SRT medium-fed seed. The 8

day seed appeared to be slightly more resistant to the toxic effects of

mercuric chloride and sodium arsenate. For the rest of the study only

the 20 day SRT seed was used.

A summary of ATA results is presented in Table 4-5. The

37

TABLE 4-3 REPRODUCIBILITY OF ATA RESULTS

Chemical Concentration Causing 50% Inhibition (mg/L)Laboratory Literature

Chloroform 0.91 0.96

Thiel et al. (1969)

Mercuric Chloride (1) 241

(2) 333

Phenol 505 500

Pearson _et a . (1980)

Sodium Arsenate (1)

(2)

10.4

6.9

38

40

30

20

10

D Control A

A Control B •

O Control C

(Medium-Fed Seed)

1

50

I

100

TIME hrs

I

150

I

200

FIGURE 4-6 REPLICATE CONTROLS - METHANE PRODUCTION

39

TABLE 4-4 COMPARISON OF 20 AND 8 DAY SRT MEDIUM-FED SEED

Chemical Concentration Causing 50% Inhibition (mg/L)20 Day Seed 8 Day Seed

Mercuric Chloride (1) 241 466

(2) 333

Sodium Arsenate (1) 10.4 11.2

(2) 6.9

40

TABLE 4-5 SUMMARY OF ATA RESULTS

Chemical Concentration Causing 50% Inhibition (mg/L)Methane Production Total Gas Production

Acetone 31,200 35,700

Beryllium Sulfate 3,340 6,440

n-Butyl Alcohol 6,690 6,730

Chloroform 0.91 1.00

Isopropyl Alcohol 31,200 26,100

Mercuric Chloride 287 -287

Methyl Isobutyl Ketone 12,900 36,400

Phenol 505 500

Potassium Chrornate 337 251

Sodium Arsenate 8.65 6.71

41

concentrations of various toxicants causing 50% inhibition of the

maximum rate of methane and total gas production are reported. Where

more than one ATA was performed, the average of computed values is

reported. In general, the concentration causing 50% inhbition of total

gas was close to the concentration causing 50% inhibition of the

maximum rate of methane production. The average percent difference

between the two values is 20%. The number of chemicals which showed

apparently greater toxicity based on total gas measurements is

approximately the same as those which showed greater toxicity based on

methane data.

4.2.2 Microtox Bioassays

Microtox bioassays were performed following the procedures

described by the manufacturer. The results of individual tests were

plotted to determine the concentration of toxicant causing 50%

inhibition of light output after 5 and 30 minutes exposure. Figure 4-7

shows typical results for a 5 minute Microtox bioassay of phenol. The

Microtox test usually provides data which show strong linearity when

plotted on semi-log graphs. The correlation coefficient for nearly all

tests was well above 0.97.

Microtox tests also show good reproducibility both between tests

and between laboratories. Table 4-6 shows the range of Microtox

5EC50fs for mercuric chloride and phenol determined in this study and

reported in the literature. Table 4-7 provides a summary of the

42

Light

Loss

70 4

60 4

50

40 4

30 -I

1.0 1.2 -1.4 1.6

Log Concentration (mg/L)

FIGURE 4-7 MICROTOX BIOASSAY - PHENOL

TABLE 4-6 REPRODUCIBILITY OF MICROTOX RESULTS

43

Mercuric Chloride

Laboratory Data

5EC50's (mg/L)

0.051

0.045

0.055

(n=3 ave=0.055 s.d.=.0041)

Literature Data 5EC50's (mg/L)

0.064 Dutka & Kwan (1981)

0.065 Bulich & Isenberg (1981)

0.08 Qureshietal. (1982)

0.07 Beckman (1983)

(n=4 ave=0.069 s.d=.0006)

Phenol

Laboratory Data

5EC50's (mg/L)

22.0

28.8

32.7

33.5

(n=4 ave=29.3 s.d.=4.55)

Literature Data 5EC50's (mg/L)

25 Lebsack (1980)

42 Samak & Noiseux (1980)

25 Bulich & Isenberg (1981)

26 Chang (1981)

28 Dutka & Kwan (1981)

40.2 Curtis (1982)

22.0 Qureshie^at. (1982)

40.7 Beckman (1983)

25 Indorato (1983)

(n=9 ave=31.1 s.d=7.80)

44

TABLE 4-7 SUMMARY OF MICROTOX RESULTS

Chemical 5EC50 (mg/L) 3QEC50 (mg/L)

Bacitracin 3,940 14,2005,556 15,503

BES * 55,500

1,2-Dichloroethylene 994 1,2901,450

Lasalocid N.T. **

Mercuric Chloride 0.051 0.0380.045 0.0310.055 0.039

Monensin N.T.

Phenol 22.028.832.733.5

30.499.969.758.6

Spiramysin N.T,

Sulfamethazine 779 672977 621706 590

Trichloroacetic acid 9,860 3,30310,600 6,250

Vinyl Acetate 2,140 2,4201,760 2,1902,170 2,640

2-Bromoethanesulfonic acidNot toxic at limit of solubility

45

Microtox results for this study.

4.2.3 Combined Results

Literature and laboratory results were combined, and are presented

in Table 4-8. The concentrations causing 50% inhibition in the 5

minute Microtox test and 50% inhibition of methane production were

compared. Figure 4-8 shows very slight correlation between these

values (r = 0.32). Approximately 45% of the chemicals were more toxic

to Microtox; 38% were more toxic to methanogens, and 17% were equally

toxic to Microtox and methanogens.

The difference between using the 5 minute and the 30 minute

Microtox results for comparison to anaerobe data was examined. Table

4-9 presents Microtox 5EC50's, 30EC50's as well as anaerobe data for

ten chemicals. The correlation coefficent for anaerobe vs. 5EC50 was

essentially the same as for anaerobe vs. 30EC50 (r= 0.41 and 0.42

respectively). These values are statistically insignificant in each

case.

4.3 COMPLEX EFFLUENTS

An initial test was made to determine the appropriate amount of

sludge-fed seed to use for ATA's. Based on the results, a 38% seed

concentration was chosen for ATA's with sludge-fed seed.

The effect of stirring on gas production was studied by comparing

stirred and non-stirred controls. Three controls with stirring bars on

46

TABLE 4-8 COMBINED DATA

Chemical Anaerobe 50% MicrotoxInhibition (mg/L) 5EC50 (mg/L)

Acetone 31,200 21,750Acrolein 11.2 0.67Acrylonitrile 212 3,910Ammonia (Free) 59 1.56Ammonium (Total) 750 4,833Bacitracin 160 4,750Beryllium Sulfate 3,340 15BES 10.5 55,500n-Butyl Alcohol 6,690 2,690Carbon Tetrachloride . 2.2 5.6Catechol 6,966 32Chloroform 0.91 6781,2-Dichloroethylene 77.5 1,220Ethyl acetate 969 1,180Formaldehyde 200 9.1Isopropyl Alcohol 31,200 42,000Mercuric Chloride 287 0.050Methyl Isobutyl Ketone 12,900 0.08Nickel Chloride 300 410Nitrobenzene 12.3 46.2Pentachlorophenol 0.5 0.08Phenol 505 29.3Potassium Chromate 337 222-Propanol 5,408 17,700Resorcinol 3,193 310Sodium Arsenate 8.65 64.5Trichloroacetic acid 340 10,200Vinyl Acetate 920 2,030Zinc Sulfate 400 29.7

47

3 -

— 9 -

1 -

U 0o

Q

a

a a

a

- 1 0 1 2 3


• Laboratory Data D Literature Data


COMBINED DATA

48

TABLE 4-9 COMPARISON OF 5 MINUTE AND 30 MINUTE MICROTOX VALUES

Chemical Anaerobe 50% Microtox Microtox

Inhibition (mg/L) 5EC50 (mg/L) 30EC50 (mg/L)

Amonia (free)

Bacitracin

1,2-Dichloroethylene

Formaldehyde

Mercuric Chloride

Nickle Chloride

Phenol

2-Propanol

Trichloroacetic Acid

Vinyl Acetate

59

160

77.5

200

287

300

505

5,408

340

920

1.56

4,750

1,220

9.1

0.050

410

29.3

17,700

10,200

2,030

2.0

14,800

1,290

3.0

0.036

13.9

64.7

35,000

4,780

2,410

49

continuously operating stirring plates were compared to regular

controls which were stirred only by shaking each time the bottles were

read. Figure 4-9 shows cumulative total gas and methane production for

the stirred and non-stirred controls. This figure shows that stirred

and non-stirred controls have similar gas production for the first few

days, after which stirred controls show both higher methane and total

gas production. The maximum rate of total gas production was 10%

higher for stirred controls. However, the maximum rate of methane

production was only 4% higher than the non-stirred controls.

The three replicates of both stirred and non-stirred controls

showed good reproducibility. Coefficients of variation were larger for

the later time points for the stirred controls, but in general ranged

between 1 and 3% for both stirred and non-stirred controls. Duplicate

ATA's of phenol and chloroform were performed. The results are in

fairly close agreement (16 and 45% differences respectivly). This is

similar to the degree of reproducibility observed for ATA's with

medium-fed sludge.

Five concentrations (2.0, 3.8, 5.7, 7.4, and 9.1%) of pulp mill

waste were tested for toxicity to methane producing bacteria using the

ATA with sludge-fed seed. Based on the results of this preliminary

test, a concentration of 3.8% PMW was chosen for evaluating combined

effects of toxicants and the pulp mill waste.

Figure 4-10 shows the results of a typical ATA run for mercuric

chloride with and without PMW. A summary of the results for complex

50

60

50

40

30

20

10

-O

A Stirred Total Gas

• Non-Stirred Total Gas

A Stirred Methane

O Non-Stirred Methane

50 100

TIME hrs

150 200

FIGURE 4-9 STIRRED AND NON-STIRRED CONTROLS (Sludge-Fed Seed)

51

40 i-

30 I-

20 H

10 h

200

A H20 Control

A PMW Control

• 130 mg/L HgCl2

O- 130 mg/L HgCl2 + .PMW

• 250 mg/L HgCl2

-Q 250 mg/L HgCl2 +

V 400 mg/L HgCl2

V 400 mg/L HgCl2 +

FIGURE 4-10 MERCURIC CHLORIDE ATA - SLUDGE-FED SEED

52

effluent studies is presented in Table 4-10. This table lists the

concentration of toxicant which results in 50% inhibition of total gas

and methane production for toxicants with and without PMW. The

concentration of toxicant predicted to cause 50% inhibition when

combined with PMW is also presented. This value is based on purely

additive effects of PMW and toxicant, and is used to determine whether

the observed combined effects are antagonistic, synergistic, or

additive. The results show that for all but one of the six tests, the

combined effect of PMW and toxicant was antagonistic based on methane

production. . For total gas production, 3 tests showed additive

interaction (observed = predicted +/- 10%); 2 showed antagonistic

interactions and one was synergistic.

The data from ATA's on chemicals without PMW using the sludge-fed

seed were compared to data using the medium-fed seed. Table 4-11

presents this information. Chloroform and mercuric chloride showed

toxicity in the same concentration range for both medium and sludge-fed

seed. Phenol and sodium arsenate were less toxic to" the sludge-fed

seed.


Data on hydrogen and carbon monoxide accumulations in toxified

anaerobic reactors were compared to Microtox data. The response

pattern of hydrogen production versus Concentration depends on the

particular toxicant. For some toxicants, hydrogen increases with

TABLE 4-10 SUMMARY OF COMPLEX EFFLUENT RESULTS

Concentration Causing 50% Inhibition of GasProduction (mg/L)

53

ToxicantTotal Gas

without with PMWMethane

without with PMW

Chloroform (1)

Chloroform (2)

Mercuric Chloride

Phenol (1)

Phenol (2)

Sodium Arsenate

0.67

1.03

255

1472

1790

82.8

0.62(0.44)*

0.94(1.03)

182(186)

1237(1488)

1861(1783)

83.2(53.2)

0.52 ' 0.50(0.43)

0.94 0.94(0.83)

223 209(187)

1774 1598(1790)

2120 2015(1589)

76.6 71.4(59.2)

* Numbers in parentheses are values predicted based on additiveeffects of PMW and toxicant.

54

TABLE 4-11 COMPARISON OF MEDIUM-FED AND SLUDGE-FED SEED

Toxicant

Chloroform

Mercuric Chloride

Phenol

Sodium Arsenate

Concentration Causing 50% Inhibition of Methane

Production (mg/L)

20 day SRTSludge-Fed

0.73 (ave)

223

1947 (ave)

76.6

20 day SRTMedium-Fed

0.91

287 (ave)

505

8.65 (ave)

8 day SRTMedium-Fed

466

11.2

55

increasing toxicant concentration, and for others it decreases. The

carbon monoxide response pattern generally is increasing carbon

monoxide production with increasing toxicant concentration

(Hickey,1987). Table 4-12 summarizes the findings of this phase of the

study. Statistical analysis of the data shows no correlation between

Microtox toxicity and hydrogen or carbon monoxide accumulations in

toxified reactors.


Prior to running tests to determine if Microtox could be used to

measure toxicity reduction by anaerobic treatment, preliminary tests

were made to determine background toxicity. Microtox tests were

performed on supernatant from anaerobic seed, air-purged supernatant,

ATA medium, and the sludge used for feed. The results of these tests

are presented in Table 4-13. These results indicate that the

background toxicity measured by Microtox is high. Air purging of the

sludge-fed seed supernatant indicated that a considerable portion of

the toxicity due to the supernatant is volatile. The sludge used for

feed showed no toxicity, suggesting that the toxicity is due to changes

that occur in the anaerobic reactor. Based on the high background

toxicities the decision was made to discontinue this phase of the

study.

4.6 SUMMARY OF RESULTS

56

TABLE 4-12 COMPARISON OF MICROTOX WITH HYDROGEN AND CARBON MONOXIDEDATA

Chemical Microtox5EC50 (mg/L)

Concentration Causing 2-Fold Changein 12 Hour Gas Production (mg/L) *

Hydrogen Carbon Monoxide

2-Bromoethane-sulfonic Acid

55,500 1,055 + 5,250 +

Chloroform

Copper

Formaldehyde

677

12.2

7.1

0.51

87.5

50

0.33 +

175

TrichloroaceticAcid

10,200

Zinc 2.5 600 50 +

* - indicates 2-fold reduction ; + indicates doubling of gasproduction.

TABLE 4-13 BACKGROUND MICROTOX TOXICITY

57

Medium-Fed Seed(Supernatant)

Sludge-Fed Seed (1)(Supernatant) (2)

5EC50

4.9%

24.0%12.9%

30EC50

23.4%20.3%

Sludge-Fed Seed(Air-Purged Supernatant)

86.8% 65.7%

ATA Medium 26.3% 25.8%

WAS Sludge(Supernatant)

Not Toxic Not Toxic

58

The initial literature review indicated that there was a fairly

good correlation between Microtox 5EC50's and concentration of toxicant

causing 50% inhibition of methane production (r = 0.74 ; n = 14).

Laboratory tests using Microtox and ATA's showed good reproducibility.

The combined laboratory and literature data showed only a slightly

significant correlation between Microtox and methane inhibition (r =

0.32 ; n = 29). The results were not changed when 30 minute Microtox

data were used instead of 5 minute data.

Comparision of ATA data using different seed for inocula showed

that sludge-fed seed was less susceptible to toxicity from certain

chemicals. For other chemicals toxicity to sludge-fed and medium-fed

seed was approximately equal. Medium-fed seed grown at 8 days SRT

instead of 20 days SRT also appeared to be somewhat more resistant to

toxicity.

Studies with a complex effluent showed predominantly antagonistic

interactions with four different chemicals. There was no statistical

relationship between Microtox data and hydrogen and carbon monoxide

accumulations in toxified anaerobic reactors. Toxicity reduction

studies were not feasible due to high background toxicity.

CHAPTER 5 -DISCUSSION

5.1 EVALUATION OF MICROTOX AS A SURROGATE FOR ATA's

A toxicity test may be useful as a surrogate for another test if

it can be shown to satisfy at least one of two criteria. The first

criterion is that a good surrogate test should have some predictable,

quantifiable relationship to the test it is to replace. The second

criterion is that the test should be as sensitive or more sensitive to

toxicants and act as a conservative estimator of toxicity. The first

criterion is best judged by statistical relationships while the second

may be judged by the percentage of toxic cases the test sucessfully

identifies, or screens out.

The initial literature survey looked promising in meeting the

second criterion. Based on the initial survey, it would seem that the

Microtox test should be able to identify methanogenic toxicants with

approximately 90% reliability. (Only 10% of the chemicals were more

toxic to methanogens.) Further study of literature values through

regression' analysis also looked promising, yielding a correlation

coefficient of 0.74. This value is close to some of the values

reported in studies comparing Microtox to other bioassays.

The laboratory data, however, did not yield such positive support

for the use of Microtox as a surrogate for the ATA test. The

correlation coeffient for this set of data was too low to reject the

null hypothesis at any level of significance (Sharp, 1979). The

59

60

laboratory data also did not indicate that Microtox would be a good

screening tool (43 % of chemicals were more toxic to methanogens). One

reason for such a discrepancy between literature and laboratory results

may be the method of choosing chemicals to test. In the literature

study, data were gathered for each test without presupposition of the

toxicity of chemicals to the other test. In the laboratory study, an

attempt was made to look at some chemicals known to be specifically

toxic to each system, some chemicals with very little toxicity to one

of the tests, and at some which were thought likely to fall in some

middle range. This deliberate testing of extremes may account for the

lack of correlation of the data. The combined data show a slight

correlation, and taken as a whole, 59 % of the chemicals studies were

of equal or greater toxicity to Microtox.

Four subsets of the combined data were evaluated: organics,

inorganics, priority pollutants and organic priority pollutants. Plots

of each data subset are presented in Figures 5-1 to 5-4. Each subset

showed insignificant correlation. Microtox seemed to be more suitable

as a screening tool for inorganic toxicants than for other the other

three groups. Only 1 of 8 inorganics (13%) was more toxic to

methanogens, compared to 29% for organics, 35% for priority pollutants

and 45% for organic priority pollutants (see Table 5-1). When

considering the types of toxicants most likely to enter an anaerobic

treatment unit, this observation is of interest. The 1980 EPA study of

priority pollutants in publicly owned treatment works (U.S. EPA, 1980)'

61

2p 4

2 -

in

3°

D

a

a

sn

a

- 1 0 1 2 3


• Laboratory Data Q Literature Data


ORGANICS

=P 4

62

-- 3.D

a

y 1 .

8

- 1 0 1 2 3

LOG MICROTOX 5EC50 (rag/L)

Laboratory Data Literature Data


INORGANICS

63

rP 4

0 H

D

a

- 1 0 1 2 3




PRIORITY POLLUTANTS

64

,*•"»

sp 4

2:O

E 3 ~is5. -i n

1 : "

in/-\

U U •

3

•

a

a

aa

f»ff

a,.-''' •

^/ a

- 1 0 1 2 3




PRIORITY ORGANICS

TABLE 5-1 STATISTICAL SUMMARY

65

Data Set

Literature Review

Literature Data

Laboratory Data

Combined Data

Organics

Inorganics

Priority Pollutants

Priority Organics

39

14

15

29

21

8

17

11

.74

insig

.32

insig

insig

insig

insig

% Data Points More Toxic toAnaerobes

10

29

43

29

13

35

45

66

lists the occurence of priority pollutants in raw sludge samples as a

percentage of times detected. Of the inorganic toxicants reported on

in both this study and the EPA study, the range of percent times

detected was from 32 to 98% with an average detection rate of 82%.

Organic toxicants, on the other hand, had a range of 2-62% with an

average detection rate of 24%.

Because the Microtox test is so short compared to the ATA, the

question arose whether a longer Microtox test might have greater

correlation to the ATA test. Ten chemicals were studied to see if 30

minute Microtox data compared more favorably than 5 minute data.

Statistical analysis showed that the two sets of data did not have any

meaningful differences.

Differences in how anaerobe toxicity was computed were also

studied. The difference in results between using methane data and

total gas data was small (20%). Based on the small difference in

results, and the increased amount of time nessecary to gather daily

methane data, using total gas as a surrogate for methane production

seems to be a reasonable alternative.

The results for this phase of the study showed unusually high

reproducibility between labs for ATA's (very much higher than within

labs). Only 2 data points were available to evaluate inter-lab

reproducibility, and due to the large number of confounding variables

expected between labs, it seems most likely that the closeness of

results is more a matter of luck than anything else.

67

5.2 COMPLEX WASTE

For the most part, the pulp mill waste showed antagonistic

interactions with the various chemicals tested. It is very difficult

to generalize from such limited data, especially when working with

poorly defined toxicants such as PMW. What can be said is that for an

anaerobic treatment unit already under stress from wastes similar to

the PMW used in this study, the additional toxic burden on the

chemicals tested would not be as great as one would otherwise suspect.

This kind of testing may have more application in a situation in which

wastes are to be combined prior to anaerobic treatment. In a case such

as this, testing for synergistic effects could help predict reactor

performance while treating mixed wastes.


No correlation could be found between Microtox 5EC50 values and

hydrogen and carbon monoxide accumulations in toxified anaerobic

reactors. The use of hydrogen and carbon monoxide monitoring is still

in the early stages of development, and the significance of these gases

as indicators of anaerobic toxicity is still being studied. The

comparison of data was furthur complicated by the variability in

response pattern seen for hydrogen accumulation for different

toxicants. While it is not the intent of this study to explore the

reasons behind this variable response, it does serve to point out the

68

complex nature of toxicity in a mixed culture such as is found in an

anaerobic reactor.


Although the toxicity reduction studies did not precede as far as

was hoped, the results do help point out an important consideration for

any treatment process. The sludge used to feed the reactor was not

toxic to Microtox. However, the effluent from the reactor (anaerobic

seed for ATA's) was toxic to Microtox, indicating that changes which

occured due to treatment resulted in toxicty. It is very likely that

the observed effect was a result of sulfate conversion to sulfide, as

evidenced in part by the volatile nature of at least some of the

toxicity. This helps to point out the sometimes conflicting goals of

waste stabilization and toxicity removal.

CHAPTER 6 - CONCLUSIONS

Overall, the results of this study do not indicate that Microtox

would be expected to serve as a particularly good surrogate for the ATA

in monitoring potentially toxic wastes entering an anaerobic treatment

unit. The initial literature review looked promising, but furthur

laboratory work reversed initial impressions. The Microtox may have an

application for monitoring waste streams which may be subject to

inorganic toxicants but are unlikely to be contaminated with organic

toxicants.

The interactive effects of a pulp mill waste and several toxicants

were evaluated. For the most part, the interactions were antagonistic.

However, generalizations to other wastes and their possible interactive

effects can not be made from the limited data available.

No correlation was observed between Microtox data and hydrogen and

carbon monoxide accumulations in toxified anaerobic reactors.

The background toxicity of the anaerobic seed used in this study

was too high to warrant continuing toxicity removal studies. It was

observed that the anaerobic treatment process itself was responsible

for some toxicity.

69

BIBLIOGRAPHY

Atkinson, D.S., Ram, N.M., and Switzenbaum, M.S., "Evaluation of the

Microtox Analyzer for Assessment of Sediment Toxicity," Technical

Report to the Massachusetts Dept. of Environmental Quality

Engineering, University of Massachusetts Environmental Engineering

Report No. 86-85-3, 1985, pp. 1-63.

Balch, W.E., Fox, G.E., Magrum, L.J., Woese, G.R., and Wolfe, R.S., "

Methanogens: Reevaluation of a Unique Biological Group,"

Microbiological Reviews, Vol. 43, 1979, pp. 260-296.

Balderston, W.L., and Payne, W.J., " Inhibition of Methanogenesis in

Salt Marsh Sediments and Whole-Cell Suspensions of Methanogenic

Bacteria by Nitrogen Oxides," Appl. Environ. Microbiol., Vol. 32,

1976, pp. 264-269.

Beckman Inc., "Microtox Application Notes: Microtox EC-50 Values,"

Beckman Instruments Inc., 1983, pp.1-6.

Beckman Inc., " Microtox System Opperating Manual," Beckman

Instruments, Inc., 1982, pp. 1-59.

Benjamin, M.M., Woods, S.L., and Ferguson, J.F., " Anaerobic Toxicity

and Biodegradability of Pulp Mill Waste Constituents," Water Res.,

Vol. 18, 1984, pp.601-607.

Bouwer, E.J., and McCarty, P.L., " Transformations of 1- and 2-Carbon

Halogenated Aliphatic Organic Compounds Under Methanogenic

Conditions," Appl. Environ. Microbiol.. Vol. 45, 1983, pp.

70

71

1286-1294.

Boyd, S.A., Shelton, D.R., Berry, D., and Tiedje, J.M., " Anaerobic

Biodegradation of Phenolic Compounds in Digested Sludge ," Appl.

Environ. Microbiol., Vol. 46, 1983, pp. 50-54.

Bulich, A.A., Greene, M.W., and Isenberg,. D.L., " Reliability of the

Bacterial Bioluminescence Bioassay for the Determination of the

Toxicity of Pure and Complex Effluents," In Aquatic Toxicology and

Hazard Assessment: Fourth Conference, D.R. Branson and K.L. Dickson

(eds.), Amer. Soc. Testing and Materials, Spec. Techn. Publ. 737,

1981, pp. 338-347.

Bulich, A.A., and Isenberg, D.L., " Use of Luminescent Bacterial System

for the Rapid Assessment of Aquatic Toxicity," Instrument soc^ Am.

Trans., Vol. 20, 1981, pp. 29-33.

Capone, D.G., Reese, D.D., and Kiene, R.P.,. " Effects of Metals on

Methanogenesis, Sulfate Reduction, Carbon Dioxide Evolution, and

Microbial Biomass in Anoxic Salt Marsh Sediments," Appl. Environ.

Microbiol.,Vol. 45, 1983, pp. 1586-1591.

Chang, J.C., Taylor, P.B., and Leach, F.R., " Use of the Microtox Assay

System for Environmental Samples," Bull. Environm. Contain. Toxicol.,

Vol. 26, 1981, pp. 150-156.

Chou, W.L., Speece, R.E., Siddiqi, R.H., and McKeon, K., " The Effect

of Petrochemical Structure on Methane Fermentation Toxicity," Prog.

Wat. Tech., Vol. 10, 1978, pp. 545-558.

Curtis, C., Lima, A., Lozano, S.J., and Veith, G.D., " Evaluation of a

72

Bacterial Bioluminescence Bioassay as a Method for Predicting Acute

Toxicity of Organic Chemicals to Fish," In Aquatic Toxicology and

Hazard Assessment: Fifth Conference, J.G. Pearson, R.B. Foster and

W.E. Bishop (eds.)i Amer. Soc. Testing and Materials, Spec. Techn.

Publ. 737, 1982, pp. 170-178.

Dutka, B.J., and Kwan, K.K., " Comparison of Three Microbial Toxicity

Screening Tests with the Microtox Test," Bull. Environ. Contam.

Toxicol. Vol. 27, 1981, pp. 753-757.

Eis, B.J., Ferguson, J.F., and Benjamin, M.M., " The Fate and Effect of

Bisulfate in Anaerobic Treatment," Journal WPCF, Vol. 55, 1983,

pp.1355-1365.

Guthrie, M.A., Kirsch, E.J., Wukasch,R.F., and Grady, C.P.L., " Fate of

Pentachlorophenol in Anaerobic Digestion," "in 1981 National

Conference on Environmental Engineering, F.M. Saunders, (Ed.),

Proceedings of the ASCE Environmental Engineering Division Specialty

Conference Atlanta, Georgia, July 8-10, 1981, published by ASCE, New

York.

Hastings, J.W., and Nealson, K.H., " Bacterial Bioluminescence," Annu.

Rev. Microbiol., Vol. 31, 1977, pp. 549-595.

Hastings, J.W., Potrikus, C.J., Gupta, S.C., Kurfurst, M., and

Makemson, J.C., " Biochemistry and Physiology of Bioluminescent

Bacteria," Adv. Microbial Physiol., Vol. 26, 1985, pp. 235-291.

Healy, J.B., and Young, L.Y., " Anaerobic Biodegradation of Eleven

Aromatic Compounds to Methane," Appl. Environ. Microbiol., Vol.38,

73

1979, pp. 84-89.

Rickey, R.F., The Role of Intermediate and Product Gases as Regulators

and Indicators of Anaerobic Digestion PhD. Dissertation, Dept.

Civil Engineering, Univ. Mass., Amherst, 1987.

Hilpert, R.t Winter, J., and Kandler, 0., " Agricultural Feed Additives

and Disinfectants as Inhibitory Factors in Anaerobic Digestion,"\

Agricultural Wastes, Vol. 10, 1984, pp. 103-116.

Hungate, R.E., " A Role Tube Method for Cultivation of Strict

Anaerobes." Meth. Microbiol., Vol. 3B, 1969, pp. 117-132.

Indorato, A.M., Snyder, K.B., and Usinowicz, P.J., " Toxicity Screening

Using Microtox," First International Symposium on Toxicity Testing

Using Bacteria, at The National Water Research Institute,

Burlington, Ontario, Canada May 1983.

Jackson, S., and Brown, V.M., " Effect of Toxic Wastes on Treatment

Processes and Watercourses," Water Poll. _Cont_., Vol. 69, 1970, p.

292.

Jarrell, K.F., and Hamilton, E.A., " Effect of Gramicidin on

Methanogenesis by Various Methanogenic Bacteria," Appl.

Environmental Microbiology, Vol. 50, 1985, pp. 179-182.

Johnson, L.D. and Young, J.C., " Inhibition of Anaerobic Digestion by

Organic Priority Pollutants ," Journal WPCF.,Vol. 55, 1983,

pp.1441-1449.

Keith, L.H., and Telliard, W.A., " Priority Pollutants I - a

Perspective View," Environmental Science and Technology, Vol. 13,

74

1979, pp. 416-423.

Lebsack, M.E., Anderson, A.D., DeGraeve, G.M., and Bergman, H,L.,

"Comparison of Bacterial Luminescence and Fish Bioassay Results for

Fossil-Fuel Process Waters and Phenolic Constituents," in Aquatic

Toxicology and Hazzard Assessment: Fourth Conference, D.R. Branson

and K.L. Dickson (eds.)i Amer. Soc. for Testing and Materials, Spec.

Techn. Publ. 737, 1981, pp. 348-356.

Lingle, J.W., and Hermann, E.R., " Mercury in Anaerobic Sludge

Digestion ," Journal WPCF..Vol. 47, 1975, pp. 466-471.

McCarty, P.L., " Anaerobic Waste Treatment Fundamentals Part Three:

Toxic Materials and their Control," Public Works, Vol. 95, 1964, pp.

91-94 .

McDermott, G.N., Moore, W.A., Post, M.A., and "Ettinger, M.B., " Copper

and Anaerobic Sludge Digestion ," Journal WPCF.. Vol. 35, 1963, pp.

655-662.

Miller,T.L., and Wolin, M.J., " A Serum Bottle Modification of the

Hungate Technique for Cultivating Obligate Anaerobes," Appl.

Microbiol., Vol. 27, 1974, pp. 985-987.

Mosey, F.E., and Hughes, D.A., " The Toxicity of Heavy Metal Ions to

Anaerobic Digestion," Wat. Pollut. Control, Vol. 74, 1975, pp.

18-33.

Nealson, K.H., and Hastings, J.W., " Bacterial Bioluminescence: Its

Control and Ecological Significance," Microbiol. Rev., Vol. 43,

1979, 496-518.

75

Neiheisel, T.W., Horning, W.B., Petrasek, A.C.,'Asberry, V.R., Jones,

D.A., Marcum, R.L., and Hall, C.T., " Effects on Toxicity of

Volatile Priority Pollutants Added to a Conventional Wastewater

Treatment System, " NTIS 1982.

Owen, W.F., Stuckey, D.C., Healy, J.B.Jr., Young, L.Y., and McCarty,

P.L., "Bioassay for Monitoring Biochemical Methane Potential and

Anaerobic Toxicity," Water Research. Vol. 13, 1979, pp.485-492.

Parkin, G.F., and Speece, R.E., "Modeling Toxicity in Methane

Fermentation Systems," J. Env. Eng. Division. ASCE , Vol 108, 1982,

pp. 515-531.

Parkin, G.F., and Speece, R.E., " Attached Verses Susppended Growth

Anarobic Reactors: Response to Toxic Substances ," Wat, jici Tech.,

Vol.15 ,1983, pp. 261-289.

Parkin, G.F., Speece, R.E., Yang, C.H.J., and Kocher, W.M., " Response

of Methane Fermentation Systems to Industrial Toxicants ," Journal

WPCF., Vol. 55, 1983, pp.44-53.

Pearson, F., Shiun-Chang, C., and Gautier, M., " Toxic Inhibition of

Anaerobic Biodegradation," Journal WFCF., Vol. 52, 1980, pp. 472-482

Peltier, W., and Weber, C.I., " Comparison of the Toxicity of Effluents

to Fish, Invertebrates and Microtox," Aquatic Biology Section

Biological Methods Branch Environmental Monitoring and Support

Laboratory U.S. Environmental Protection Agency Cincinnati, Ohio

July, 1980.

76

Qureshi, A.A., Flood, K.W., Thomson, S.R., Janhurst, S.M., Inniss,

C.S.t and Rokosh, D.A., " Comparison of a Luminescent Bacterial Test

with Other Bioassays for Determining Toxicity of Pure Compounds and

Complex Effluents," In Aquatic Toxicology and Hazard Assessment:

Fifth Conference, J.G. Pearson, R.B. Foster and W.E. Bishop (eds.)»

Amer. Soc. Testing and Materials, Spec. Techn. Publ. 737, 1982, pp.

179-195 .

Ribo, J.M., and Kaiser, K.L.E., " Effects of Selected Chemicals to

Photoluminescent Bacteria and their Correlations with Acute and

Subleathal Effects on Other Organisms," Chemosphere. Vol. 12, 1983,

pp. 1421-1442.

Russell, L.L., Cain, C.B., and Jenkins, D.I., " Impact of Priority

Pollutants on Publicly Owned Treated Works "Processes: A Literature

Review ," Proceedings of the 37th Industrial Waste Conference,

Purdue University, 1982, pp. 871-883.

Samak, Q.M., and Noiseux, R., " Acute Toxicity Measurement by the

Beckman Microtox," Presented at the Seventh Annual Aquatic Toxicity

Workshop in Montreal, Canada, November, 1980.

Sellers, K.E., Studies on the Actions and Interactions of Heavy Metals

on Bioluminescent Bacteria. Thesis, Dept. Civil Engineering, Univ.

Mass., Amherst, 1985.

Sharp, V.F., Statistics for the Social Sciences, Little, Brown and Co.,

Boston, 1979, pp. 381.

Sheehan, K.C., Sellers, K.E., and Ram, N.M., " Establishment of a

77

Microtox Laboratory and Presentation of Several Case Studies Using

Microtox Data," Technical Report to the Massachusetts Dept. of

Environmental Quality Engineering, University of Massachusetts,

Engineering Reopt No. 77-83-8, 1984, pp. 1-76.

Shelton, D.R., and Tiedje, J.M.,"" General Method for Determining

Anaerobic Biodegradation Potential," Appl. Environ. Microbiol., Vol.

48, 1984, pp. 840-848.

Slattery, G.H., " Biomonitoring and Toxicity Testing : Toxicant

Treatability Definition and Measurement," Presented at the WPCF

Analytical Techniques in Water Pollution Control Conference,

Cininnati, Ohio, May 2-3, 1985.

Smith, M.R., and Mah, R.A., " Growth and Methanogenesis by

Methanosarcina Strain 227 on Acetate and Methanol ," Appl. Environ.

Microbiol..Vol. 36, 1978, pp. 870-879 .

Speece, R.E., "Anaerobic Biotechnology for Industrial Wastwater

Treatment," Environ. Sci. Technology, Vol. 17, 1983, pp. 416A-427A.

Speece, R.E., and Parkin, G,F., "The Response of Methane Bacteria to

Toxicity," Proceedings Third International Symposium on. Anaerobic

Digestion, Boston Massachusetts, 14-19 August, 1983.

Stuckey, D.C., Owen, W.F., McCarty, P.L., and Parkin, G.F., " Anaerobic

Toxicity Evaluation by Batch and Semi-Continuous Assays," Journal

WPCF, Vol. 52, 1980, pp.720-729.

Sullivan, D.E., and Lambert, W.P., " Development of a Short Term

Anaerobic Digestion Effects Test," Presented at the WPCF 56th

78

Conference, Industrial Waste Symposia, Atlanta, Georgia, Oct. 3-7,

1983.

Switzenbaum, M.S., "Anaerobic Treatment of Wastewater: Recent

Developments." ASM News, Vol. 49, 1983, pp. 532-536.

Sykes, R.M., and Kirsch, E.J., " Accumulation of Methanogenic

Substrates in CC14 Inhibited Anaerobic Sewage Sludge Digester

Cultures ," Water Res., Vol. 6, 1972, pp. 41-55 .

Taylor, G.T., "The Methanogenic Bacteria," Progress in Industrial

Microbiology, (Bull, M., Editor), Vol. 16, 1982, pp. 231-329.

Thiel, P.G., " The Effect of Methane Analogues on Methanogenesis in

Anaerobic Digestion ," Water Res,, Vol. 3, 1969, pp. 215-223 .

United States Environmental Protection Agency, "Fate of Priority

Pollutants in Publicly Owned Treatment Works : Interim Report, " EPA

- 440/1-80-301, 1980, pp. 1-180.

Varel, V.H., and Hashimoto, A.G., " Methane Production by Fermentor

Cultures Acclimated to Waste from Cattle Fed Monensin, Lasalocid,

Salinomycin, or Avoparcin ," Appl_._ Environ. Microbiol., Vol. 44,

1982, pp. 1415-1420.

Vasseur, P., Ferard, J.F., Vail, J., and Larbaight, G., "Comparison of

Bacterial Luminescence and Daphnia Bioassay Results for Industrial

Wastewaters Toxicity Assessment, " Environmental Pollution, Vol. 34,

1984, pp. 225-235

Vogel, T.M., and McCarty, P.L., "Biotransformation of

Tetrachloroethylene to Trichloroethylene, Dichloroethylene, Vinyl

79

Chloride, and Carbon Dioxide under Methanogenic Conditions," Appl.

Environ. Microfaiol., Vol. 49, 1985, pp. 1080-1083.

Wang, Y.T., Suidan, M.T., and Pfeffer, J.T., "Anaerobic Biodegradation

of Indole to Methane," Appl. Environ. Microbiol., Vol. 48, 1984, pp.

1058-1060.

Yang, J., Speece, R.E., Parkin, G.F., Gossett, J., and Kocher, W., "

The Response of Methane Fermentation to Cyanide and Chloroform,"

Prog. Wat. Technol., Vol. 12, 1980, pp. 977-989.

Zinder, S.H., Anguish, T., and Cardwell, S.C., "Selective Inhibition by

2-Bromoethanesulfonate of Methanogenesis for Acetate in a

Thermophilic Anaerobic Digester," Appl, Environ._ Microbiol., Vol 47,

1984, pp. 1343-1345.

assessment of the microtox bioassay as a preditor for anaerobic bacterial toxicity ·...

Documents